ST104P, an anti-angiogenic agent

ABSTRACT

The anti-angiogenic functions of a polysulfated, cyclic compound, ST104P, were investigated. ST104P exhibited excellent water solubility and low cytotoxicity to endothelial cells. ST104P potently inhibited the secretion of matrix metalloproteinase (MMPs) by endothelial cells. Moreover, ST104 also perturbed the migration and tube formation of endothelial cells. Application of ST104P abolished the neovascularization in chicken choroiallantoic membrane (CAM) in a dose-dependent manner. Besides, repeated administration of ST104P into Lewis lung carcinoma resulted in delayed tumor growth and prolonged the life span of tumor-bearing mice. These results indicated that ST104P inhibited angiogenesis and may hold promises for treatment of cancer and diseases or conditions caused by excessive angiogenesis.

FIELD OF THE INVENTION

This invention relates to pharmacology and medicine. In particular, itrelates to a method and a pharmaceutical compound exhibiting remarkableanti-angiogenic activity useful for cancer therapy as well as fordiseases or conditions caused by excessive angiogenesis.

BACKGROUND OF INVENTION

Angiogenesis, the recruitment of new blood vessels, is an essentialcomponent of the metastatic pathway (Zetter, B. R., Annu Rev Med, 49:407-424 (1998)). Persistent, unregulated angiogenesis occurs in amultiplicity of disease states, tumor metastasis and abnormal growth byendothelial cells and supports the pathological damage seen in theseconditions. These vessels provide the principal route by which tumorcells exit the primary tumor site and enter the circulation. For manytumors, the vascular density can provide a prognostic indicator ofmetastatic potential, with the highly vascular primary tumors having ahigher incidence of metastasis than poorly vascular tumors. Tumorangiogenesis is regulated by the production of angiogenic stimulatorsincluding members of the fibroblast growth factor and vascularendothelial growth factor families. In addition, tumors may activateangiogenic inhibitors such as angiostatin and endostatin that canmodulate angiogenesis both at the primary site and at downstream sitesof metastasis. The potential use of these and other natural andsynthetic angiogenic inhibitors as anticancer drugs is currently underintense investigation. Such agents may have reduced toxicity and be lesslikely to generate drug resistance than conventional cytotoxic drugs.

In addition, angiogenesis has been associated with blood-born tumorssuch as leukemias, any of various acute or chronic neoplastic diseasesof the bone marrow in which unrestrained proliferation of white bloodcells occurs, usually accompanied by anemia, impaired blood clotting,and enlargement of the lymph nodes, liver, and spleen. It is believedthat angiogenesis plays a role in the abnormalities in the bone marrowthat give rise to leukemia-like tumors.

Excessive angiogenesis or abnormal growth of new blood vessels has alsocontributed to a variety of other diseases or conditions that arecommonly known in the art to be associated with or otherwise mediated byangiogenesis. One such example is ocular neovascular disease. Thisdisease is characterized by invasion of new blood vessels into thestructures of the eye such as the retina or cornea. In age-relatedmacular degeneration, the associated visual problems are caused by aningrowth of chorioidal capillaries through defects in Bruch's membranewith proliferation of fibrovascular tissue beneath the retinal pigmentepithelium. Angiogenic damage is also associated with diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma and retrolental fibroplasias. Other diseases ormedical conditions associated with angiogenesis known in the artinclude, but without limitation to, rheumatoid arthritis, systemiclupus, polyarteritis, sickle cell anemia, osteoarthritis, veinocclusion, artery occlusion, carotid obstructive disease andatherosclerosis. Therapies directed at control of the angiogenic processcould lead to the abrogation or mitigation of these diseases andconditions. Clinical trials are now underway to develop optimumtreatment strategies for antiangiogenic agents (U.S. Pat. No.6,518,298).

ST104P {(tetrameric cyclic compound of 4,5-dihydroxynaphthalene-2,7,disulfonic acid linked by methylene bridges (Poh B.-L., et al, Tetra.Letters 30(8):1005-1008 (1989), Poh, B.-L., and Lim, C. S., Tetrahedron46(10):3651-3658 (1990a), and Poh, B.-L., et al, Tetrahedron46(12):4679-4386 (1990b)) is a synthetic polysulfated-, cyclo-,tetrachromotropylene macrocyclic compound containing four naphthaleneunits in its cyclic structure. It is a water-soluble compound withmarginal cellular toxicity. The functions of ST104P as anti-viral agentsand anti-thrombotic treatment have been indicated in previous studies(U.S. Pat. Nos. 5,166,173, 5,196,452, 5,312,837, 5,441,983 and5,409,959). However, ST104P has never been indicated to be ananti-angiogenic agent exhibiting anti-angiogenic functions useful fortreatment of various diseases or conditions caused by or in associationwith undesirable angiogenesis including, but not limited to, cancertherapy.

It is theoretically possible that a compound exhibiting anti-viral andanti-thrombotic activity may also possess other biological activitieseither by a similar or an entirely different mechanism. It is thereforean object of the present invention to provide for a compositioncomprising ST104P exhibiting a remarkable anti-angiogenic activitysuitable for cancer therapy.

It is a further object of the invention to provide for a method ofproviding therapeutical benefits for a human patient in cancer therapyemploying a composition comprising ST104P

It is a further object of the invention to provide for a compositioncomprising ST104P exhibiting a remarkable activity against diseases orconditions mediated by or in connection with angiogenesis including,without limitation, diabetic retinopathy, age-related maculardegeneration, rheumatoid arthritis, osteoarthritis, atherosclerosis,corneal neovascularization, retinal/choroidal neovascularization andsystemic lupus.

SUMMARY OF THE INVENTION

This invention relates to an anti-angiogenic function of a polysulfated,cyclic compound, ST104P, that exhibits angiogenesis inhibition in vitroand in vivo without overt cytotoxicity. ST104P exhibits excellent watersolubility and low cytotoxicity to endothelial cells while potentlyinhibited the secretion of matrix metalloproteinases (MMPs) byendothelial cells. Moreover, ST104 also perturbed the migration and tubeformation of endothelial cells. Application of ST104P abolished theneovascularization in chicken chorioallantoic membrane (CAM) in adose-dependent manner. Furthermore, repeated administration of ST104Pinto Lewis lung carcinoma resulted in delayed tumor growth and prolongedthe life span of tumor-bearing mice. These results indicated that ST104Pinhibited angiogenesis and is useful for treatment of cancer and otherdiseases and conditions mediated by angiogenesis.

One aspect of the invention is to provide for an anti-tumor agent fortreating tumors in a human or animal by the inhibition of angiogenesisin said tumors comprising a tetrameric cyclic compound of4,5-dihydroxynaphthalene-2,7, disulfonic acid linked by methylenebridges in a pharmaceutical acceptable carrier. The pharmaceuticalacceptable carrier may be adapted for oral, sublingual, rectal, vaginal,nasal or parenteral (intravenous, intraperitoneal, intratumor)administration. Additionally, the pharmaceutical acceptable carrier maybe adapted in the form of a tablet, a capsule, a cachet, a solution, anemulsion, a depository, or a powder.

Another aspect of the invention is to provide for a an angiogenesisinhibitor for inhibiting angiogenesis in tumors in a human or animalcomprising a tetrameric cyclic compound of 4,5-dihydroxynaphthalene-2,7,disulfonic acid linked by methylene bridges in a pharmaceuticalacceptable carrier. The pharmaceutical acceptable carrier may be adaptedfor oral, sublingual, rectal, vaginal, nasal or parenteral (intravenous,intraperitoneal, intratumor) administration. Additionally, thepharmaceutical acceptable carrier may be adapted in the form of atablet, a capsule, a cachet, a solution, an emulsion, a depository, or apowder.

One more aspect of the invention is to provide for an angiogenesisinhibitor for treating a non-tumor condition or disease associated withangiogenesis in a human or animal comprising a tetrameric cycliccompound of 4,5-dihydroxynaphthalene-2,7, disulfonic acid linked bymethylene bridges in a pharmaceutical acceptable carrier. Theundesirable condition or disease associated with angiogenesis accordingto the present invention includes, but not limited to, polyarteritis,sickle cell anemia, vein occlusion, artery occlusion, carotidobstructive disease, atherosclerosis, rheumatoid arthritis, systemiclupus, and osteoarthritis. The pharmaceutical acceptable carrier usedhereto in connection with treatment of a non-tumor condition or diseaseassociated with angiogenesis may be adapted for oral, sublingual,rectal, vaginal, nasal, transdermal, ophthalmic (intravitreal,intracameral) or parenteral administration. Additionally, thepharmaceutical acceptable carrier may be adapted in the form of atablet, a capsule, a cachet, a solution, an emulsion, a depository, apatch, or a powder.

One further aspect of the invention is to provide for a method oftreating tumors in a human or animal by the inhibition of angiogenesisin said tumors, which comprises administering thereto an effectiveangiogenesis inhibiting dose of a tetrameric cyclic compound of4,5-dihydroxynaphthalene-2,7, disulfonic acid linked by methylenebridges in a pharmaceutical acceptable carrier. The pharmaceuticalacceptable carrier used hereto in connection with treatment of tumorsassociated with angiogenesis may be adapted for oral, sublingual,rectal, vaginal, nasal or parenteral (intravenous, intraperitoneal,intratumor) administration. Additionally, the pharmaceutical acceptablecarrier may be adapted in the form of a tablet, a capsule, a cachet, asolution, an emulsion, a depository, or a powder.

One further aspect of the invention is to provide for a method ofinhibiting angiogenesis in tumors in a human or animal comprisingadministering thereto an effective angiogenesis inhibiting dose of atetrameric cyclic compound of 4,5-dihydroxynaphthalene-2,7, disulfonicacid linked by methylene bridges in a pharmaceutical acceptable carrierthat may be adapted for oral, sublingual, rectal, vaginal, nasal orparenteral (intravenous, intraperitoneal, intratumor) administration.Additionally, the pharmaceutical acceptable carrier may be adapted inthe form of a tablet, a capsule, a cachet, a solution, an emulsion, adepository, or a powder.

One further aspect of the invention is to provide for a method oftreating a non-tumor condition or disease associated with angiogenesisin a human or animal which comprises administering thereto an effectiveangiogenesis inhibiting dose of a tetrameric cyclic compound of4,5-dihydroxynaphthalene-2,7, disulfonic acid linked by methylenebridges in a pharmaceutical acceptable carrier. According to the presentinvention, the undesirable condition or disease associated withangiogenesis that may be benefited from the method includes, but notlimited to, diabetic retinopathy, age-related macular degeneration,polyarteritis, sickle cell anemia, vein occlusion, artery occlusion,carotid obstructive disease, atherosclerosis, rheumatoid arthritis,systemic lupus, and osteoarthritis.

The pharmaceutical acceptable carrier used hereto in connection withtreatment of a non-tumor condition or disease associated withangiogenesis may be adapted for oral, sublingual, ophthalmic (includingintravitreal or intracameral), rectal, vaginal, nasal, transdermal, orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by conventionalpharmaceutical techniques. Such techniques include the step of bringinginto association the active ingredient and the pharmaceutical carries(s)or excipient(s). Additionally, the pharmaceutical formulations adaptedby the invention suitable for oral administration may be presented asdiscrete units in the form of a tablet, a capsule, a cachet, a solution,an emulsion, a depository, a patch, or a powder each containing apredetermined amount of the active ingredient, etc. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The cytotoxicity of ST104P in different types of cells. (A)ST104P altered the morphologies of endothelial cells but not 3T3 cells.After treatment of ST104P at 10-500 μg/ml in DMEM medium containing 10%FCS for 24 h, the morphologies of BAEC and 3T3 cells were monitoredunder phase contrast microscopy. The pictures were taken at400×magnification. (B). The dose-dependent effect of ST104P on theproliferation of different cells. After treatment with ST104P at 10-500μg/ml or PBS for 48 h, the viability of various cells was determined bycrystal violet method in an ELISA reader. Each data represent mean±SD ofquadruplicate experiments. Asterisks indicated statistic significanceversus control (P<0.001).

FIG. 2. ST104P inhibited the migration of endothelial cells. The effectof ST104P on migration of endothelial cells. BAEC were plated in 6-wellplate (1×10⁶ cells per well) and treated with vasostatin of indicateddose for 12 h. After trypsinization, endothelial cells were applied totop wells in Boyden chamber to initiated migration towardchemoattractant bFGF (100 ng/ml) in the bottom wells. The migrated cellson filter were stained and counted. Each point represents mean±SEM oftriplicate experiments.

FIG. 3. ST104P abolished the tube formation of endothelial cells.Application of ST104P perturbed tubular network of BAEC or EA.hy9236cells in Matrigel. Endothelial cells were applied to Matrigel-coatedplate in the absence or presence of ST104P. After 8 h, the tubularstructures of BAEC cells were monitored and recorded under lightmicroscopy. The pictures were taken at 400×magnification.

FIG. 4 shows the effect of ST104P on MMPs secretion by endothelialcells. ST104P decreases the MMPs secretion by endothelial cells. Theconditioned media of BAECs or EA.hy926 cells (in 24-well plate at 1×10⁴cells/well) treated with PBS or ST104P (10-500 μg/ml) for 24 h werecollected to analyze the expression of MMPs by gelatin-PAGE zymography.

FIG. 5 shows the effect of ST104P on neovascularization in chickenembryos. The CAMs in 8-day old chicken embryos were incubated withsaline or ST104P (50 or 200 μg in saline) for 48 h. The angiogenesisprofiles on CAMs were recorded under dissection microscope. There wasactive and intact blood vessels formation in PBS-treated CAM. Incontrast, the vascular network was severely destroyed in ST104P-treatedCAMs.

FIG. 6. Injection of ST104P suppressed tumor growth of Lewis lungcarcinoma in mice. The subcutaneous dorsa of mice were implanted withLewis lung carcinomas. The tumor sizes in mice during treatment withST104P or control were recorded. Treatment was performed by intratumorinjection of ST104P (100 μg in 0.1 ml PBS) daily from day 0 to day 10 asindicated by horizontal bar. Each point represents mean±SEM for tenmice. The experiment was repeated with comparable results.

FIG. 7. Injection of ST104P prolonged the survival of tumor-bearingmice. The subcutaneous dorsa of mice were implanted with Lewis lungcarcinomas. The tumor sizes in mice during treatment with ST104P orcontrol were recorded. Treatment was performed by intratumor injectionof ST104P (100 μg in 0.1 ml PBS) daily from day 0 to day 10 as indicatedby horizontal bar. Each point represents mean±SEM for ten mice. Theoverall survival rates of ten mice in PBS— or ST104P-treated group wererecorded. Kaplan-Meier survival analysis indicated the ST104P treatmentsignificantly enhanced the survival rates of tumor-bearing mice(P<0.001). The experiment was repeated with comparable results.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods

Reagents

Recombinant basic fibroblast growth factor (bFGF) was purchased from R&DSystem (Minneapolis, Minn.). Matrigel was from BD PharMingen (La Jolla,Calif.). The stock solution of ST104P is prepared in normal saline orphosphate buffered saline at 1 mg/ml or at such other concentrations orin such other different formulations as deemed desirable and appropriateaccording to the purposes of the intended assays.

Cell Culture

Human umbilical vein endothelial cells (HUVEC; passage 3 to 6) wereisolated from umbilical veins and cultured in RPMI 1640 medium (LifeTechnologies, Gaithersburg, Md.) containing 15% fetal calf serum, 20U/ml porcine heparin (Sigma Chemical Co), and 100 μg/ml endothelial cellgrowth supplement (Calbiochem; La Jolla, Calif.). Bovine aorticendothelial (BAEC) cells, 3T3 cells, and LL2 Lewis lung carcinoma cellswere cultured with DMEM (Dulbecco's Modified Eagle Medium; Gibco BRL,Rockville, Md.) containing 10% fetal calf serum (PAA, Austria), 2 mMglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL,Rockville, Md.) in 5% CO₂ at 37° C. Human vascular endothelial EA.hy926cells were cultured in DMEM (Dulbecco's Modified Eagle Medium; GibcoBRL, Rockville, Md.) containing 10% fetal calf serum (PAA, Austria), 100μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT; GibcoBRL, Rockville, Md.), 2 mM glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin (Gibco BRL, Rockville, Md.) in 5% CO₂ at 37° C.

Cell Proliferation Assay

The effect of ST104P on the viability of various cells was determinedusing crystal violet stain assay. Briefly, cells cultured in 96-wellplate (2-4×10³ cells/well) were treated with various doses of ST104P for48 h. After treatment, cells were fixed with 2.5% glutaraldehyde at roomtemperature for 15 minutes, stained with 0.1% crystal violet solution(in 20% methanol; 20 μl per well) for 20 minutes, washed with distilledwater for three times, solublized with solution containing 50% ethanoland 0.1% acetic acid. The dye in viable cells was measured by readingthe optical density at 590 nm using a scanning multi-wellspectrophotometer (ELISA reader; Dynatech Laboratories, Chantilly, Va.).

Cell Migration Assay

Endothelial cells were treated with ST104P of indicated doses for 6-12h, harvested by trysinization, collected by centrifugation, resuspendedin DMEM media containing 0.1% BSA, and seeded in triplicate for eachdose and controls in the upper compartment of the chamber (1.2×10⁵ cellsin 400 μl). The lower compartment was filled with 200 μl of the DMEMmedia containing 100 ng/ml bFGF (R&D, Minneapolis, Minn.) as thechemoattractant, or with DMEM media containing 0.1% BSA as the negativecontrol (to evaluate random migration). The compartments were separatedby a polycarbonate filter (8-μm pore size; Nucleopore, Costar,Chambridge, Mass.) coated with 0.005% gelatin to allow cell adhesion.After incubation for 2-4 h in a humidified 5% CO₂ atmosphere at 37° C.,cells on the upper side of the filter were removed, and those that hadmigrated to the lower side were fixed in absolute ethanol, stained with10% Giemsa solution (Merck, Germany), and counted as a mean±SD perfilter under five different high power fields.

Tube Formation Assay

Matrigel (Becton Dickinson; Bedford, Mass.) was diluted with coldserum-free medium to 10 mg/ml. Two hundred μl of the diluted solutionwere added to each well in 24-well plate and allowed to form a gel at37° C. for 30 min. BAEC or HUVEC (1.5×10⁵ cells per ml) were initiallyincubated for 15 min with various doses of ST104P in complete medium.Two hundred μl of the cell suspension (3×10⁴ cells) were thensubsequently added to each well and incubated for 6-8 h at 37° C. in 5%CO₂. Under these conditions endothelial cells form delicate networks oftubes that are detectable within 2-3 h and are fully developed after8-12 h. After incubation with ST104P, the wells were washed and theMatrigel and its endothelial tubes were fixed with 3% paraformaldehyde.

Matrix Metalloproteinases (MMPs) Zymography

Secretion of MMPs by endothelial cells was assessed by 0.1%gelatin-SDS-PAGE zymography. Briefly, endothelial cells near 80%confluence were washed twice with serum-free media and treated withvarious doses of vasostatin for 24-48 h. Conditioned media werecollected and assayed for protein concentration by Bradford assay.Aliquots of conditioned media were subjected to separation with 10%SDS-PAGE containing 0.1% type A gelatin (Sigma; St. Louis, Mo.). Afterelectrophoresis, gel was washed twice with 2.5% Triton X-100, incubatedin buffer containing 40 mM Tris-HCl, pH 8.0, 10 mM CaCl₂, 0.01% sodiumazide at 37° C. for 12-24 h, stained with 0.25% Coomassie Blue R-250 in50% methanol and 10% acetic acid for 1 h, and destained with 10% aceticacid, 20% methanol. The gelatinolytic regions by MMPs were visualized aswhite bands in blue background.

Chorioallantoic Membrane (CAM) Assay

The CAM assay was carried out as described previously (Sheu et al.,Anticancer Res 18, 4435-4441,1998). Briefly, fertilized White Leghornchicken eggs were purchased from Taiwan Provincial Research Institutefor Animal Health (Hsin-Chu, Taiwan) and incubated at 37° C. with 60%humidity. On day 3, a square window was opened in the shell, and 2 to 3ml of albumen was removed to allow detachment of the developing CAM. Thewindow was sealed with a glass, and the eggs were returned to theincubator. On day 8, 50-100 μg ST104P in saline was injected onto thetop of the CAM. The CAMs were examined daily until day 10-12, when theangiogenic response peaks, and photographed in ovo with astereomicroscope equipped with Zeiss MC63 camera system (Zeiss,Oberkochen, Germany). The experiments were carried out using 10 eggs pergroup. In the presence of angiogenesis inhibitors, massive loss ofvascular structure occurred and leaded to mortality of chicken embryos,which was used as another index to evaluate the extent of angiogenesisinhibition.

Animal Studies

Animal study was carried out in the animal facility of KaohsiungVeterans General Hospital in accordance with institutional guidelines.Male C57BL/6J mice (6- to 8-week-old; Animal Center of National ChengKung University, Taiwan) were used. Mice were acclimated and caged ingroups of four or less. All mice were fed with a diet of animal chow andwater ad libitum. Animals were anesthetized in a methoxyflurane chamberprior to all procedures and were observed until fully recovered. Animalswere sacrificed by a lethal dose of methoxyflurane.

Treatment of Lewis Lung Carcinoma

C57BL/6J mice were implanted with Lewis lung carcinomas cells using thetechniques described previously (O'Reilly et al., Cell 88,277-285,1997). Briefly, the LL2 Lewis lung carcinoma cells wereresuspended in PBS at the concentration of 2.5×10⁶ cells per ml andinjected into C57B16/J mice (n=20) with 0.1 ml of the cell suspension toinduce tumor. Tumors were measured with a dial-caliper and volumes weredetermined using the formula width²×length×0.52. After tumor volumesreached 100-200 mm³, mice were randomized into two groups receivingperiodic injection of ST104P (n=10) or saline (n=10) by subcutaneousinjection at a site near tumor. The measurement of tumor size wasterminated when mice began to die. In addition, the survival rate ofmice in different groups was recorded and analyzed by Kaplan-MierSurvival analysis.

Results

Low Cytotoxicity of ST104P in Different Types of Cells

We investigated the effects of ST104P in endothelial cells includingBAEC, HUVEC, EA.hy926 cells, and in non-endothelial cells including 3T3cells, GH3 cells, C6 cells, HepG2 cells, SK-Hep-1 cells, MDCK cells.Morphological analysis indicated that ST104P are not very toxic to alltypes of cells (data not shown). Application of ST104P moderatelyinhibited the proliferation in different types of cells in adose-dependent manner (FIG. 1). However, ST104P treatment failed toelicit significant cytotoxicity that the IC₅₀ for ST104P in BAEC,EA.hy926, and 3T3 cells were much larger than 500 μg/ml (˜350 μM; FIG.1)

ST104P Inhibited the Migration of Endothelial Cells

To analyze the effect of ST104P on migration of endothelial cells, an invitro migration system in Boyden chamber was exploited with bFGF aschemoattractant. After incubation with endothelial cells for 6 h, ST104Ptreatment prominently inhibited the migration of endothelial cell towardchemoattractant bFGF in a dose-dependent manner (IC₅₀ 100 μg/ml for BAECand EA.hy926; FIG. 2)

ST104P Perturbed the Tube Formation of Endothelial Cells

The abilities of endothelial cells to form tube-like structure in thepresence of ST104P were also investigated. Application of ST104P (20-100μg/ml) effectively abolished the vessel-like structure of BAEC orEA.hy926 cells in Matrigel (FIGS. 3A and 3B)

Tube Formation MMP Secretion

Angiogenesis can be divided into several distinct steps including matrixmetalloproteinases (MMPs) secretion, proliferation and migration ofendothelial cells. Thus, we examined the effects of ST104P on theseangiogenic processes in BAEC and EA.hy926 cells. MMPs, a family ofzinc-containing endopeptidase, mediate selective degradation ofextracellular matrix that is required for migration and invasion ofendothelial cells at the onset of angiogenesis. To determine the effectof ST104P on MMPs secretion, the conditioned media from ST104P-treatedendothelial cells were subjected to gelatin-zymography analysis. ST104Ppotently inhibited the secretion of MMP-2 and MMP-9 by endothelial cellseven at dose as low as 10 μg/ml (FIG. 4). These data suggested thatST104P may affect the de novo synthesis or release of MMPs inendothelial cells.

ST104P Inhibited Angiogenesis Chicken Chorioallantoic Membrane

To study the ability of ST104P in inhibiting angiogenesis in vivo, weused the CAM assay in 8-day old chick embryos because extensiveneovascularization occurs during that period. At a dose of 50 μg perCAM, there was 50% inhibition of angiogenesis in ST104P-tretaed CAM,whereas, PBS had no obvious effects on neovasularization in CAMs nor onthe mortality rate of chicken embryos (FIG. 5). At an escalated dose of200 μg per CAM, the extensive inhibition of neovascularization by ST104Pled to 90% mortality of chick embryos (9 out of 10 died inST104P-treated groups versus 0 in PBS-treated group). These dataindicated that ST104P abolished the neovascularization during chickenembryo development.

Repeated Injection of ST104P Suppressed the Tumor Growth in Mice

We treated established Lewis lung carcinoma grown in syngeneic C57BL/6Jmice by periodic intratumor injection of ST104P into tumors. The growthof Lewis lung primary tumors was suppressed by subcutaneous injection ofST104P (100 μg) for four times that the average tumor size ofST104P-treated mice (2620±320 mm³) was significantly smaller (˜40%decrease) than that of saline-treated groups (4876±670 mm³; P<0.05; FIG.6). In addition, mice treated with ST104P survived significantly longerthan animals of vehicle-treated group (P<0.01; FIG. 7). There was noevident weight loss or adverse effects in mice treated with ST104Psuggesting that ST104P injection was well tolerated by animals.Together, these results indicate that ST104P may be applicable to cancertherapy.

DISCUSSION

In the present study, we demonstrate that ST104P is a potent inhibitorfor MMP secretion and formation of tube-like structure in endothelialcells. The inhibitory mechanism of ST104P in CAM angiogenesis and tumorgrowth seems to be mediated via its anti-angiogenic effects. Unlikeother angiogenesis inhibitor such as TNP-470 (Moulton et al.,Circulation 99, 1726-1732, 1999), angiostatin (O'Reilly et al., Cell 79,315-328,1994), or endostatin (O'Reilly et al., Cell 88, 277-285,1997),ST104P does not directly inhibit the proliferation of endothelial cells.Instead, ST104P indirectly suppressed angiogenesis via blockade of otherangiogenesis steps such as migration, tube formation, and MMP secretion.In contrast to TNP-470 or endostatin, ST104P is soluble and couldadministrated via saline or buffer systems. Recently, human clinicaltrials on MMP inhibitors have been completed and yielded differentialoutcomes. Because of the high potency in halting MMP secretion, ST104Pmay enter clinical trials as based o its indication as MMP inhibitor.

Because of its low cytotoxicity, ST104P treatment was well tolerated byanimals via various routes including oral or intravenous,intraperitoneal, and intratumor injection without overt signs of adverseeffects (data not shown). Future studies on the toxicology,pharmokinetics and biodistribution of ST104P are warranted to gainfurther insights in the safety and side effects of ST104P. Since ST104Pis an aromatic compound, the carcinogenic potential should beextensively evaluated during pre-clinical trial. In summary, ST104P is asoluble angiogenesis inhibitor with low cytotoxicity that may holdpotential for future clinical application.

Other diseases or conditions that are commonly known in the art to bemediated by angiogenesis and which can be treated by an angiogenesisinhibitor such as ST104P according to the invention are as follows:

Atherosclerosis

Neovascularization within the intima of human atherosclerotic lesions iswell described. In normal vessels, the microvascular network of vasavasorum is confined to the adventitia and other media. However, invessels with atherosclerotic involvement, these networks become moreabundant and extended to the intima of atherosclerotic lesions. Plaquevessels are often found in areas rich in macrophages T cells and mastcells, which cell types are known to activate angiogenesis. One of themain factors associated with atherosclerosis is oxidized low-densitylipoproteins (LDL), which also causes apoptosis of endothelial cells. Ithas been demonstrated that endostatin is beneficial to endothelial cellgrowth exposed to mildly oxidized LDL and significantly reduceatherosclerosis in genetically susceptible mice (Ren et al, Methods FindExp Clin Pharmacol, 24(4): 159-199; 2002). In addition, prolongedtreatment with angiogenesis inhibitors such as endostatin or TNP-470reduced plague growth and intimal neovascularization in apolipoproteinE-deficient mice (Moulton et al., Circulation; 99: 1726-1732, 1999)

Rheumatoid Arthritis

Inflammatory joint diseases such as rheumatoid arthritis (RA) are amajor cause of disability and are frequently associated with increasedmorbidity and mortality. During the rheumatoid arthritis there isenlargement and increased cellularity of the synovial lining of joints,before invasion by the synovium of the underlying cartilage and bone.The increased tissue mass requires a network of blood vessels to supplynutrients and oxygen. Disruption of synovial angiogenesis is thus adesirable aim of antiarthritic therapies. It has been shown that theangiogenesis inhibitor, protease-activated kringles 1-5, reduces theseverity of murine collagen-induced arthritis. The clinical efficacy ofthis treatment was reflected by a reduction in joint inflammation anddestruction, which further suggests that antiangiogenic therapies thatblock formation of new blood vessels and reduce synovial expansion canbe effective in treating rheumatoid arthritis (Sumariwalla et al,Arthritis Research and Therapy; 15(1) 32-39, 2002). In addition,systemic administration of endostatin in passive murine collagen inducedarthritis exhibits inhibition of arthritis by inhibiting pannusformation and bone destruction (Kurosaka et al, Ann Rheum Dis, 62;677-697, 2003).

Systemic Lupus

Agent exhibiting anti-inflammatory and immunomodulatory activity alsoshows activity in treatment of systemic lupus (Alfadley et al., J AmAcad Dermatol, 48(5) S89-S91, 2003; Housman et al, Arch Dermatol,139:50-54, 2003; Ossandon et al, Clinical and Experimental Rheumatology,20:709-718, 2002; U.S. Pat. No. 6,518,298) Cutaneous manifestations oflupus erythematosus are chronic, disfiguring lesions that may beassociated with systemic diseases. These cutaneous lesions can be simplyclassified into 3 categories: (1) vascular lesions seen in systemiclupus erythematosus; (2) interface lesions seen in chronic cutaneouslupus erythematosus; or (3) special lesions as seen in lupuserythematosus profundus or in vesiculobullous lesions. It has beenreported that thalidomide, an anti-inflammatory agent and animmunomodulator exhibiting anti-angiogenic activity, provides dramaticimprovement to patients with systemic as well as cutaneous lupuserythematosus. (Alfadley et al., J Am Acad Dermatol, 48(5) S89-S91,2003; Housman et al, Arch Dermatol, 139:50-54, 2003; Ossandon et al,Clinical and Experimental Rheumatology 20:709-718, 2002; U.S. Pat. No.6,518,298).

Osteoarthritis

Angiogenesis may also have a role in osteoarthritis. Destruction of thejoint is shown to be caused by the activation of the chondrocytes byangiogenic-related factors, which would subsequently promote new boneformation. Accordingly, therapeutic intervention that prevents the bonedestruction could stop the progress of the disease and provide relieffor persons suffering with arthritis (Smith J O, et al., J. Orthop Sci.,8: 849-857, 2003).

Age-Related Macular Degeneration

Age-related macular degeneration (ARMD) is a major cause of acquiredblindness, which results from development of choroidalneovascularization associated with overlying retinal damages. Choroidalneovascularization is also generated in high myopia, angioid streaks,and some inflammatory diseases. Angiostatin, a known inhibitor of vesselendothelial proliferation in vitro and vessel growth inside tumors, isalso shown to significantly reduce the sizes of choroidalneovascularization lesion (Lai et al, Investigative Ophthalmology andvessel Science, vol. 42 (10): 2401-2407, 2001)

Diabetic Retinopathy

Antiangiogenic agents have been clinically studied in the treatment ofretinal disease for patient with central retinal vein occlusion, branchretinal vein occlusion and diabetic retinopathy. For instances, vascularendothelial growth factor (VEGF), a mediator for angiogenesis in manyexperimental and clinical situation, is also a potent “vascularpermeability factor” in inducing leakage of blood vessels. Evidence hasaccumulated linking upregulation of VEGF with both increasedpermeability of ocular blood vessels and the development ofneovascularization in the eye. In monkey eyes, vascular leakage, likediabetic retinopathy, can be induced by injection of VEGF. And indiabetic animals and diabetic patients, there appears to be an earlyupregulation of VEGF, which can be responsible for the development ofthe retinopathy including neovascularization. Thus, agents that inhibitVEGF are potential therapeutic agents for both diabetic retinopathy andage-related macular degeneration. (Jampol, L. M., Disclosure of AmericanAcademy of Ophthalmology 2002 Annual Meeting; October 18-9, Orlando,Fla.; Hans-Peter Hammes; Ocular Complications in Diabetes, 59^(th)Annual Scientific Sessions of ADA, San Diego, Calif. 1999)

Retinal/Choroidal Neovascularization

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum,systemic lupus erythematosis, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, mycobacterial infectionsetc. Other diseases include, but are not limited to, diseases associatedwith rubeosis (neovascularization of the angle) and diseases caused bythe abnormal proliferation of fibrovascular or fibrous tissue includingall forms of proliferative vitreoretinopathy (U.S. Pat. No. 6,518,298.)

All referenced patents, applications and literatures are incorporatedherein by reference in their entirety. Furthermore, where a definitionor use of a term in a reference, which is incorporated by referenceherein is inconsistent or contrary to the definition of that termprovided herein, the definition of that term provided herein applies andthe definition of that term in the reference does not apply. It shouldbe apparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refer to at leastone of something selected from the group consisting of A, B, C . . . .and N, the text should be interpreted as requiring only one element fromthe group, not A plus N, or B plus N, etc.

1. A method of treating tumors by inhibition of angiogenesis in saidtumors comprising administration to an animal in need of such treatmentwith an effective angiogenesis inhibiting dose of a compound having theformula,

in a pharmaceutically acceptable carrier.
 2. The method of treatingtumors of claim 1 wherein the pharmaceutically acceptable carrier isadapted for oral, sublingual, rectal, nasal or parenteraladministration.
 3. The method of treating tumors of claim 1 wherein thepharmaceutically acceptable carrier is adapted in a form of a tablet, acapsule, a cachet, a solution, an emulsion, or a powder.
 4. A method ofinhibiting angiogenesis in tumors by administering to an animal in needof such treatment with an effective angiogenesis inhibiting dose of acompound having the formula,

in a pharmaceutically acceptable carrier.
 5. The method of inhibitingangiogenesis of claim 4 wherein the pharmaceutically acceptable carrieris adapted for oral, sublingual, rectal, nasal or parenteraladministration.
 6. The method of inhibiting angiogenesis of claim 4wherein the pharmaceutically acceptable carrier is adapted in a form ofa tablet, a capsule, a cachet, a solution, an emulsion, or a powder. 7.The method of treating tumors of claim 1, wherein the animal is a human.8. The method of inhibiting angiogenesis of claim 4, wherein the animalis a human.